The broad objective of this project is to elucidate how lens fiber cells, their membranes, cytoskeleton and interlocking junctions all interact with each other to maintain the transparency of the organ. Lens fibers possess an elaborate interlocking system and adherens junctions (AJs) for maintaining their structural stability, especially during the deformation that accompanies visual accommodation. Gap junctions are needed for the exchange of ions and small metabolites between avascular lens cells. Also, aquaporin-0 constitutes 50% of total integral fiber-cell membrane proteins, and as an adhesion molecule, helps maintain the narrowed extracellular space between fiber cells to minimize light scattering. A number of mutations of AQP0 are directly associated with various forms of cataracts in humans and animals. For the first time, our recent data have shown that AQP0, highly enriched in protrusions, plays an unconventional role in maintaining the normal structure of these domains. With loss of AQP0, protrusions specifically undergo uncontrolled elongation and breakdown, leading to fiber-cell separation and cataract. By using wild-type, AQP0-/- and AQP0+/- mice, Aim 1 will demonstrate a novel cooperative role played by N-cadherin-based AJs in regulating AQP0-rich protrusions for maintaining the stability of protrusions. We will analyze cell-cell adhesion formations of AQP0 and cadherin proteins expressed in adhesion-deficient mouse fibroblast L-cells. We rationalize that AQPO-based protrusions maintain an overall stability of fiber cells at the gross level, while N-cadherin-based AJs support localized adhesions in protrusions and between fiber cells at the microscopic level. Ball-and-sockets (B&S) and protrusions are two distinct interlocking domains in lens fibers. The goal of Aim 2 is to demonstrate that AQP0 plays a role in regulating gap junction-rich B&S domains during fiber cell differentiation. We will also test the mechanistic role of AQP0 in regulating Cx50 and Cx46 in B&S domains in Cx50-KO, Cx46-KO and double KO lenses. Aim 3 will identify the functional role of an actin-based cytoskeletal organization in maintaining a normal structure of protrusions. We will identify the actin-based structural organization and its associated proteins, and determine the biochemical associations that exist between actin, cytoskeletal proteins, and AQP0-binding proteins with membranes of the mouse lenses. The proposed work will employ state-of-the-art techniques such as our improved whole-mount preparations, 3-D ultrastructural visualization, high-resolution freeze-fracture immunogold labeling, TEM, SEM, and biochemical analysis. Impact: Successful completion of the three aims will provide for the first time a novel mechanistic view of: (1) a cooperative adhesion role played by both AQP0-based protrusions and N-cadherin-based AJs in maintaining the stability and transparency of the lens, (2) the crucial role played by AQP0 via maintaining a normal structure of gap junction-rich B&S domains in facilitating cell-to-cell communications in metabolically active fiber cells, and (3) ho AQP0 interacts with underlying actin-based cytoskeleton complexes to control the shape and function of interlocking domains.